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. 2019 Jun 18;10(3):e00886-19. doi: 10.1128/mBio.00886-19

FIG 3.

FIG 3

LpxEAA complements E. coli strains deficient in C55-PP phosphatase or PGP phosphatase activities. (A) Complementation of the C55-PP phosphatase-deficient E. coli strain (BW25113 ΔybjG ΔpgpB ΔbacA::kan) by the temperature-sensitive pMAK705 plasmid harboring bacAEC (positive control, pMAK-bacAEC) or lpxEAA (pMAK-lpxEAA). WT E. coli cells carrying pMAK705 or C55-PP phosphatase-deficient E. coli cells carrying pMAK-bacAEC or pMAK-lpxEAA were grown at 30°C or 42°C. From left to right are spots of 10-fold serial dilutions from 102 to 105. (B) Complementation of the PGP phosphatase-deficient E. coli strain (W3110 ΔpgpA ΔpgpB ΔpgpC::kan) by the temperature-sensitive pMAK705 plasmid harboring pgpAEC (positive control, pMAK-pgpAEC) or lpxEAA (pMAK-lpxEAA). WT E. coli cells carrying pMAK705 or PGP phosphatase-deficient E. coli cells carrying pMAK-pgpAEC or pMAK-lpxEAA were grown at 30°C or 42°C. From left to right are spots of 10-fold serial dilutions from 102 to 105. (C) Chromosomal complementation of C55-PP phosphatase activity-deficient E. coli with lpxEAA. The left, middle, and right images show the construction of different E. coli C55-PP phosphatase gene deletion mutants, the growth of WT E. coli cells and C55-PP phosphatase-deficient cells complemented by a chromosomal copy of lpxEAA, and the PCR verification of ybjG, bacA, and pgpB knockouts of the target mutant strain, respectively. (D) Chromosomal complementation of PGP phosphatase activity-deficient E. coli with lpxEAA. The left, middle, and right images show the construction of different E. coli PGP phosphatase gene deletion mutants, the growth of WT E. coli cells and PGP phosphatase-deficient cells complemented by a chromosomal copy of lpxEAA, and PCR verification of pgpA, pgpC, and pgpB knockouts of the target mutant strain, respectively. Since the expected sizes of pgpB (1,106 bp) and pgpB::PL-lpxEAA (1,093 bp) are similar using primers flanking pgpB in the final strain, the knockout of pgpB was established by also verifying the PCR result of the mother strain (strain 5: W3110 ΔpgpA ΔpgpB::PL-lpxEAA-frt-kan-frt).